Unexplained Microcytosis Research Articles

εγ-Thalassemia, a New Hemoglobinopathy Category.

Large β-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or β-, δβ-, γδβ-, and ϵγδβ-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for β-thalassemia. Notably, ϵγδβ-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψβ loci with intact LCR, δ-, and β-regions in 2 women and newborn twins. Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψβ (HBBP1) loci. This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδβ-thalassemia.

English

BACKGROUND Among microcytic hypochromic anaemias, the most common disorders are iron deficiency anaemia (IDA) and co-pathological conditions such as α- or βthalassemia (α- or β- thalassemia) traits. Thalassemia minor is often an asymptomatic carrier state but exhibit marked microcytosis that can be mistaken for iron deficiency. About 1.5 % of the global population (80 to 90 million people) are carriers of thalassemia and more than 200 mutations are described in thalassemia, the only effective way to reduce burden of thalassemia is to prevent birth of homozygotes. The purpose of this study was to estimate the prevalence of beta thalassemia trait (BTT) in adult individuals with microcytic hypochromic anaemia and among antenatal mothers visiting the Obstetric Department of SDMCMSH. METHODS In this prospective descriptive study, 101 adult patients between 18 and 40 years who were in in-patient department (IPD) or attending out-patient department (OPD) from department of general medicine and department of haematology were studied during a period of one year from October 2018 to October 2019 and their blood sample was sent for complete blood count (CBC), iron studies and haemoglobin electrophoresis. All the data was entered in Microsoft Excel software and analysis was done using Minitab software. P value of less than 0.05 was considered statistically significant. RESULTS Out of the 101 microcytic hypochromic anaemia patients analysed, 7 patients have beta thalassemia trait. 4 of the 7 are females. Their haemoglobin electrophoresis report showed high HBA2 value of > 3.8. There were no carriers among the pregnant individuals tested. A regression model was fitted for predicting HBA2 based on values of serum iron, serum ferritin, and mean corpuscular volume (MCV). CONCLUSIONS In conclusion, identification of thalassemia gene mutations in our population is necessary because of the country’s multiracial population. This will prevent unnecessary treatment with iron supplements in thalassaemic patients and also helps in pregnant women to prevent thalassemia major children. Molecular genotyping provides a rapid and reliable method for identification of common, and unknown α - and β - gene mutations, which help to diagnose unexplained microcytosis and thus prevent unnecessary iron supplementation. We conclude that microcytosis due to thalassemia is common in north Karnataka. This illustrates the importance of adequate prenatal and laboratory investigation for these abnormalities. KEYWORDS Beta Thalassemia Trait, Microcytosis, Haemoglobin Electrophoresis, Antenatal Screening, Thalassemia

Microcytosis as a risk marker of cancer in primary care: a cohort study using electronic patient records.

Microcytosis (smaller than normal red blood cells) has previously been identified as a possible early risk marker for some cancers. However, the role of microcytosis across all cancers has not been fully investigated. To examine cancer incidence in a cohort of patients with microcytosis, with and without accompanying anaemia. Cohort study of patients aged ≥40 years using UK primary care electronic patient records. The 1-year cancer incidence was compared between cohorts of patients with a mean red cell volume of <85 femtolitres (fL) (low) or 85-101 fL (normal). Further analyses examined sex, age group, cancer site, and haemoglobin values. Of 12 289 patients with microcytosis, 497 had a new cancer diagnosis within 1 year (4.0%, 95% confidence interval [CI] = 3.7 to 4.4), compared with 1465 of 73 150 without microcytosis (2.0%, CI = 1.9 to 2.1). In males, 298 out of 4800 with microcytosis were diagnosed with cancer (6.2%, CI = 5.5 to 6.9), compared with 940 out of 34 653 without (2.7%, CI = 2.5 to 2.9). In females with microcytosis, 199 out of 7489 were diagnosed with cancer (2.7%, CI = 2.3 to 3.1), compared with 525 out of 38 497 without (1.4%, CI = 1.3 to 1.5). In patients with microcytosis but normal haemoglobin, 86 out of 2637 males (3.3%, CI = 2.6 to 4.0) and 101 out of 5055 females (2.0%, CI = 1.6 to 2.4) were diagnosed with cancer. Microcytosis is a predictor of underlying cancer even if haemoglobin is normal. Although a benign explanation is more likely, clinicians in primary care should consider simple testing for cancer on encountering unexplained microcytosis, particularly in males.

Alpha-thalassaemia trait as a cause of unexplained microcytosis in a South African population

To the Editor: I read the article by Loonat et al. with interest. In the early 1980s a study was undertaken at Red Cross War Memorial Children's Hospital, Cape Town, South Africa (SA), to assess the frequency with which a low red cell mean corpuscular volume (MCV) was associated with the presence of thalassaemia or an abnormal haemoglobin. Between January 1979 and December 1980, 730 patients with an MCV of ≤60 were investigated. Forty-six (6.4%) were found to carry a η-thalassaemia gene and 20 (2.7%) had an abnormal haemoglobin, most commonly Hb E. The prevalence of thalassaemia was highest in individuals of mixed ethnic origin, and abnormal haemoglobins were found exclusively in this population, although the numbers of white and black patients were much smaller in comparison. Alpha-thalassaemia was not tested for, as the technology was unavailable at the time owing to cost constraints. Nevertheless the findings confirmed that patients with persistent unexplained microcytosis/hypochromia should be screened for thalassaemia and haemoglobin variants.

Alpha-thalassaemia trait as a cause of unexplained microcytosis in a South African population.

Red cell microcytosis is a common abnormality detected in a full blood count, which often prompts clinicians to investigate further for a cause. In the absence of iron deficiency and anaemia of chronic disease, the differential diagnosis includes β-thalassaemia trait and α-thalassaemia trait. We investigated the contribution of α-thalassaemia trait in South African subjects with unexplained microcytosis. Iron studies, haemoglobin subfractionation and multiplex polymerase chain reaction (PCR) analysis for α-globin gene deletions were performed on 97 controls and 86 patients. After excluding iron deficiency, anaemia of chronic disease and β-thalassaemia trait, 78.0% of subjects with unexplained microcytosis were confirmed on PCR analysis to have α-thalassaemia trait. Alpha-thalassaemia trait accounts for the majority of unexplained microcytosis.

Incidence of ATRX mutations in myelodysplastic syndromes, the value of microcytosis.

Acquired α-thalassemia myelodysplastic syndrome (MDS) (ATMDS) is an acquired syndrome characterized by a somatic point mutation or splicing defect in the ATRX gene in patients with myeloid disorders, primarily MDS. In a large MDS patient series, the incidence of ATMDS was below 0.5%. But no large series has yet assessed the incidence of ATMDS in microcytic MDS. In this study, we focused on patients with MDS and unexplained microcytosis, which was defined as absence of iron deficiency, inflammatory disease, or history of inherited hemoglobinopathy. Our data confirm the low frequency of ATRX mutations in MDS: 0% in an unselected clinical trial cohort of 80 low risk MDS, 0.2-0.8% in a multicenter registry of 2,980 MDS and 43% of MDS with unexplained microcytosis in this same registry. In addition, we reported four novel mutations of the ATRX gene in ATMDS. This study further determines the frequency of ATRX mutations and highlights the importance of microcytosis to detect ATRX mutations within MDS patients.

A novel double heterozygous Hb Fontainebleau/HbD Punjab hemoglobinopathy

ObjectivesTo report the finding of a novel double heterozygous hemoglobinopathy, the coinheritance of Hb Fontainebleau (α-chain variant) with HbD-Punjab (β-chain variant) discovered upon investigation of unexplained microcytosis in an infant. Design and methodsHemoglobinopathy investigation was performed by high performance liquid chromatography (HPLC) using the β-thalassemia Short Program on the Bio-Rad Variant IITM followed by gel electrophoresis at alkaline and acid pH (Sebia Hydrasys 2 Electrophoresis System) and molecular diagnostic testing. This study complied with our institutional board ethics requirements. ResultsHPLC and electrophoresis suggested a complex α- and β-chain hemoglobinopathy with presumptive identification of the beta Hb variant as Hb D-Punjab. DNA sequencing analysis revealed the presence of a double heterozygous status for Hb Fontainebleau/Hb D-Punjab. ConclusionsIn this paper we report the coinheritance of Hb Fontainebleau with Hb D-Punjab.

Molecular Characterization of Hb Hamilton Hill (HBA2: c.388delC), a Novel HBA2 Variant Generating a Premature Termination Codon and Truncated HBA2 Chain

In recent years, the identification of α-thalassemias caused by nondeletional mutations has increased significantly due to the advancement of sensitive molecular genetics tools. We report clinical and experimental data for a novel frameshift mutation caused by a single base deletion at position 388 in exon 3 of the α2-globin gene (HBA2: c.388delC; Hb Hamilton Hill), resulting in the phenotype of α-thalassemia (α-thal). Hb Hamilton Hill was identified in an adult female of unknown ethnicity investigated for unexplained microcytosis. Direct DNA sequencing of the HBA2 gene revealed a heterozygous mutation, HBA2: c.388delC, and the molecular effect of this mutation was assessed experimentally using our previously described in vitro model. The experimental analysis involved transfection of a human bladder carcinoma (5637) cell line with expression vectors carrying either HBA2-wild type (HBA2-WT) or HBA2: c.388delC followed by total RNA purification and cDNA synthesis. Both wild type and mutant gene expression was studied and compared at the transcriptional and translational levels using quantitative real time polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC), respectively. Our experimental data showed a significant reduction by 25.0% (p = 0.04) in the transcriptional activity generated from HBA2: c.388delC compared to HBA2-WT. As a result of this base deletion, a frameshift in the open reading frame generates a premature termination codon (PTC) at codon 132 of exon 3 resulting in the formation of a truncated α-globin chain. The truncated α-globin chain, observed by the IFC technique, is most likely unstable and undergoes a rapid turnover resulting in the thalassemic phenotype.

Incidence of Atrx Mutations in Myelodysplastic Syndromes (MDS)

INTRODUCTIONAcquired α-thalassemia MDS (ATMDS) is an acquired syndrome characterized by a somatic point mutation or splicing defect in the ATRX gene in patients with myeloid disorders, primarily MDS. No large series have assessed its incidence in these pathologies. The ATRX protein is a chromatin-remodeling factor that may act as a transcriptional cofactor and plays an important role in the epigenetic regulation of gene expression (1). The unusual microcytosis in the context of MDS is due to a reduced synthesis of α-globin related to a dramatic down regulation of α-globin gene expression by inactivating mutation in the ATRX gene (2). We tried to better determine the frequency of ATRXmutations in a MDS registry and a clinical trial cohort.METHODSWe first used next-generation sequencing (NGS) on samples from 80 patients included in GFM-AZA-EPO-2008-01 study, a phase II study of azacitidine combined to EPO in IPSS low and int-1 risk MDS patients resistant to EPO. All coding exons of ATRXwere sequenced using the following methodology: Library preparation was performed using the Ion Ampliseq™ Library kit 2.0-384 LV (Life Technologies®). Template-positive Ion Sphere Particles™ were prepared using the Ion OneTouch™ 200 Template Kit v2 (Life Technologies®). Sequencing was performed with the Ion PGM™ 200 Sequencing Kit and the Ion 318™ Chip on the Personal Genome Machine® (PGM™) platform (Life Technologies®). Data files were independently analysed using two softwares: Torrent Browser™ (Life Technologies®) and SeqNext™ (JSI® Medical Systems).We then focused on the GFM registry, started in 2003, which includes 2980 MDS patients (at diagnosis regardless of the MDS type) and searched by direct Sanger method for mutations in the ATRXgene in MDS with unexplained microcytosis (MCV lower or equal to 80), ie without iron deficiency, inflammatory disease or history of inherited hemoglobinopathy.RESULTSNGS of ATRX gene in the 80 patients included in GFM-AZA-EPO-2008-01 study found no mutation. Of the 2980 patients in the GFM register, 139 had MCV below 80, unexplained in 58 of them. Out of these 58 patients, 14 had available material for ATRX gene sequencing by Sanger method. 2/14 patients were already known to have ATMDS, a RCMD and a CMML. In the remaining 12 patients, a mutation was found in 4 cases: a missense mutation in exon 9, a missense mutation in exon 4, a deletion in intron 9 with a frame shift and an addition in exon 1. Those mutations, to our knowledge, had not yet been described in ATMDS.Therefore, of 2980 patients with MDS, we described 6 ATMDS (5 males, 1 female), ie an incidence of 6/2980 or 0.2%. However, 6/14 or 43% of patients with unexplained microcytosis had a mutation of ATRXand only 14/58 patients with unexplained microcytosis could be sequenced, suggesting an incidence closer to 6x58/14/2980 or 0.8%. There was no recurrent mutation. Of the 6 patients (median age was 76 years), 1 had RCUD, 1 CMML, 1 RAEB-2 and 3 RAEB-1. All karyotypes were different: favorable (n=2), intermediate (n=3) and poor (n=1) and IPSS was low (n=1), int-1 (n=3) and high (n=1). Median survival was 31.5 months. In the CMML patient, microcytosis was found 7 years before diagnosis. In the other 5 patients, microcytosis was discovered concomitantly with MDS. In 4 patients, microcytosis disappeared during the disease course. 2 patients died from AML progression, 2 from other causes and 2 were still alive.CONCLUSIONOur data confirm the low frequency of ATRX mutations in MDS: 0% in an unselected clinical trial cohort of 80 lower risk MDS, 0.2 to 0.8% in a multicenter registry of 2980 MDS and 42% of MDS with unexplained microcytosis in this same registry. We also confirmed that these mutations can be found in all types of MDS. Larger studies are required to determine the prognostic value of these rare and non recurrent ATRXmutations. 1.Law MJ. Cell. 2010.2.Steensma DP, Blood. 2005 DisclosuresNo relevant conflicts of interest to declare.

Genetic basis of persistent red blood cell microcytosis in the Chinese unexplained by phenotypical testing

AimsHypochromic microcytic anaemia is the hallmark phenotype of thalassaemia. Current phenotypical tests do not provide a diagnosis in a small proportion of patients with red blood cell microcytosis. We aim...

The Spectrum of α-Thalassemia Mutations in the Kurdish Population of Northeastern Iraq

In an attempt to determine the spectrum of α-thalassemia (α-thal) mutations in the Kurdish population of Northeastern (NE) Iraq, a total of 101 unrelated adults with unexplained hypochromia and/or microcytosis were enrolled. α-Thalasssemia mutations were characterized by gap polymerase chain reaction (gap-PCR), multiplex PCR (m-PCR) and reverse hybridization and sequencing for both α genes. A total of nine α-thal mutations were characterized including four deletional ones: –α3.7 (rightward), – –MED-I, –(α)20.5, –α4.2 (leftward) and five nondeletional ones: αpolyA1α, ααAdana, α−5 ntα, αCSα and αpolyA2α. These determinants were arranged in 12 different genotypes, the most frequent of which were: –α3.7/αα, – –MED-I/αα, –α3.7/–α3.7, αpolyA1α/αα, ααAdana/αα and –(α)20.5/αα. This pattern is similar to that reported in Turkey, western (W) Iran, Cyprus and Greece, and to some extent, different from the pattern observed in the Arabian Peninsula.

Molecular and Cellular Characterization of a New α-Thalassemia Mutation (HBA2:c.94A>C) Generating an Alternative Splice Site and a Premature Stop Codon

The identification of α-thalassemia (α-thal) due to point mutations has been increasing significantly with the advancement of molecular diagnostic tools. We describe here the molecular and cellular characteristics of the thalassemia mutation HBA2:c.94A>C, a novel point mutation affecting the α2-globin gene, causing a mild α-thal phenotype in a male patient of undisclosed ethnicity, investigated for unexplained microcytosis. The detected mutation is located at the penultimate nucleotide (nt) of the first exon which we postulated might affect pre mRNA splicing. While an in silico analysis did not predict any aberrant splice variants, experimental analysis using our in vitro model for gene expression studies showed utilization of a cryptic splice site at codon 15 that resulted in an aberrant splice variant. As a result, a frameshift in the reading frame of the mature mRNA was produced, leading to the formation of a premature termination codon (PTC) between codons 48 and 49 in exon 2. This in turn leads to nonsense mediated mRNA decay (NMD) and the phenotype of α-thal.

Significant haemoglobinopathies: guidelines for screening and diagnosis

Antenatal screening/testing of pregnant women should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. Newborn screening and, when necessary, follow up testing and referral, should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. All babies under 1 year of age arriving in the UK should be offered screening for sickle cell disease. Preoperative screening for sickle cell disease should be carried out in patients from ethnic groups in which there is a significant prevalence of the condition. Emergency screening with sickle solubility tests must always be followed by definitive analysis. Laboratories performing antenatal screening should utilize methods capable of detecting significant variants and be capable of quantitating haemoglobins A 2 and F at the cut-off points required by the national antenatal screening programme. The laboratory must ensure a provisional report is available within three working days from sample receipt.

Molecular Characterization of α-Thalassemia in the Dohuk Region of Iraq

The molecular basis of α-thalassemia (α-thal) has been addressed by several studies from the eastern Mediterranean region, but not from Iraq. To address this issue, we studied 51 individuals with unexplained hypochromia and/or microcytosis, as well as nine patients with documented Hb H disease from the Dohuk region in northern Iraq. We used multiplex gap-polymerase chain reaction (gap-PCR), reverse hybridization, and sequencing for this purpose. It was found that the most common genotypes in those with unexplained hypochromia and/or microcytosis were −α3.7/αα, followed by − −MED-I/αα, then −α3.7/−α 3.7, respectively, detected in 84.3% of the above individuals. Other genotypes identified sporadically were −α4.2/αα, αpoly A1α/αα (AATAAA>AATAAG), αAdanaα/αα [Hb Adana, codon 59 (Gly→Asp) or HBA1:c.179G>A], and αEvanstonα/αα [Hb Evanston, codon 14 (Trp→Arg) or HBA1:c.43 T>C]. Three cases (5.88%) remained uncharacterized even after sequencing. All nine Hb H cases carried the −α3.7/− −MED-I genotype. Such findings are rather different from those in other eastern Mediterranean populations, particularly with relevance to an Hb H molecular basis.

Genotyping of alpha-thalassemia in microcytic hypochromic anemia patients from North India

Microcytic hypochromic anemia is a common condition in clinical practice and alpha-thalassemia has to be considered as a differential diagnosis. Molecular diagnosis of alpha-thalassemia is possible by polymerase chain reaction. The aim of this study was to evaluate the frequency of alpha-gene numbers in subjects with microcytosis. In total, 276 subjects with microcytic hypochromic anemia [MCV<80fl; MCH<27pg] were studied. These include 125 with thalassemia trait, 48 with thalassemia major, 26 with sickle-cell thalassemia, 15 with E beta-thalassemia, 40 with iron-deficiency anemia, 8 with another hemolytic anemia, and 14 patients with no definite diagnosis. Genotyping for -alpha3.7 deletion, -alpha4.2 deletion, Hb Constant Spring, and a-triplications was done with polymerase chain reaction. The overall frequency of -alpha3.7 deletion in 276 individuals is 12.7%. The calculated allele frequency for a-thalassemia is 0.09. The subgroup analysis showed that co-inheritance of a-deletion is more frequent with the sickle-cell mutation than in other groups. We were able to diagnose 1/3 of unexplained cases of microcytosis as a-thalassemia carriers. The a-gene mutation is quite common in the Indian subcontinent. Molecular genotyping of a-thalassemia helps to diagnose unexplained microcytosis, and thus prevents unnecessary iron supplementation.

Prevalence of α-Globin Gene Deletions Among Patients with Unexplained Microcytosis in a North-American Population

Increasing multi-ethnicity is likely to make α-thalassemia (α-thal) more prevalent in Western metropolitan areas. Multiplex polymerase chain reaction (m-PCR) allows rapid and precise identification of most of α-thal carriers. With this method, we sought to determine the prevalence of α-thal and the corresponding genotype, among all non repetitive consecutive blood samples that had an unexplained microcytosis. These specimens had been sent to the hematology laboratory for a blood count analysis, found to be microcytic, and secondarily tested for ferritin level and hemoglobin (Hb) high performance liquid chromatography (HPLC) profile. Five hundred and sixteen microcytic blood samples were evaluated and 197 samples with normal ferritin and Hb HPLC were studied by m-PCR. Among 196 interpretable PCRs, 48 α-thal cases (24.5%) were identified: 28 with a single α-globin gene deletion and 20 with two α-globin gene deletions. Of these 20 cases, six showed two deletions in cis. None of the erythrocytic parameters studied predicted the presence of α-thal deletions. We conclude that a significant proportion (24.5%) of blood counts with microcytosis not explained by an iron deficiency, an inflammatory state or an abnormal Hb on HPLC, are caused by an α-globin gene deletion. The pertinence of genetic counseling for α-thal based on molecular diagnosis should be evaluated more formally in urban centers where this genetic condition is likely to have an increasing prevalence and clinical relevance.

Prevalence of α-Globin Gene Deletions Among Patients with Unexplained Microcytosis in a North-American Population

Increasing multi-ethnicity is likely to make alpha-thalassemia (alpha-thal) more prevalent in Western metropolitan areas. Multiplex polymerase chain reaction (m-PCR) allows rapid and precise identification of most of alpha-thal carriers. With this method, we sought to determine the prevalence of alpha-thal and the corresponding genotype, among all non repetitive consecutive blood samples that had an unexplained microcytosis. These specimens had been sent to the hematology laboratory for a blood count analysis, found to be microcytic, and secondarily tested for ferritin level and hemoglobin (Hb) high performance liquid chromatography (HPLC) profile. Five hundred and sixteen microcytic blood samples were evaluated and 197 samples with normal ferritin and Hb HPLC were studied by m-PCR. Among 196 interpretable PCRs, 48 alpha-thal cases (24.5%) were identified: 28 with a single alpha-globin gene deletion and 20 with two alpha-globin gene deletions. Of these 20 cases, six showed two deletions in cis. None of the erythrocytic parameters studied predicted the presence of alpha-thal deletions. We conclude that a significant proportion (24.5%) of blood counts with microcytosis not explained by an iron deficiency, an inflammatory state or an abnormal Hb on HPLC, are caused by an alpha-globin gene deletion. The pertinence of genetic counseling for alpha-thal based on molecular diagnosis should be evaluated more formally in urban centers where this genetic condition is likely to have an increasing prevalence and clinical relevance.

Prevalence of Alpha-Globin Gene Deletions amongst Patients with Unexplained Microcytosis in a North-American Population.

Introduction: Increasing multi-ethnicity is likely to make α-thalassemia more prevalent in western metropolitan areas. The current prevalence of α-thalassemia in regions outside the traditional thalassemia areas with the associated risk for HbH disease and hydrops foetalis is unknown. Genetic counseling for reproductive risks due to α-globin gene mutations requires adequate and precise genotyping.Objectives: To determine by multiplex polymerase chain reaction (m-PCR) the prevalence and genotypes of α-thalassemia among non selected, consecutive cases of unexplained microcytosis identified in a clinical hematology laboratory. To evaluate if differences or variations in erythrocytic indices are indicative of α-thalassemia.Methods: During a period of 3 months, α-globin genotype was determined by m-PCR on all non repetitive microcytic (MCV <80fL) blood samples from adults with normal ferritin and normal hemoglobin HPLC analysis (Variant I, Bio-Rad). A previously described m-PCR analysis allowed the detection of α-globin gene deletions and specifically identified each of the following 7 deletions when present: −α3.7, −α4.2, −−SEA, −−FIL, −−MED, −−THAI and −α(20.5).Results: 516 microcytic blood samples were evaluated of which 197 had normal ferritin and Hb HPLC analysis and were submitted for m-PCR. Among 196 interpretable m-PCRs, 148 did not have α-globin gene deletion(s) (group 1) and 48 α-thalassemia cases (24,5%) were identified: 28 cases of single α-globin gene deletion (group 2) and 20 cases of 2 α-globin gene deletions (group 3). In group 3, 6 cases showed deletions in cis. Results on differences between the groups are presented in Table 1. Even though some differences were statistically different, none was clinically useful and diagnostic.Conclusion: A significant proportion (24,5%) of cases with microcytosis not likely explained by iron deficiency, inflammation or a hemoglobinopathy are caused by α-globin gene deletion(s). This laboratory survey, despite a short period of sample collection (3 months) identified 6 carriers of a 2 gene deletion in cis that may have a severe impact on reproductive decisions and potential future utilisation of resources. These findings are likely to be reproducible in other North American cosmopolitan cities where α-thalassemia is not endemic but likely to be of increasing prevalence due to immigration. The availability, precision and reliability of diagnostic methods such as m-PCR and the increasing ethnic diversity of North-American cities point to an emerging need to develop genetic counseling programs based on the molecular diagnosis of α-thalassemia.Erythrocytic parameters with their standard deviation in the different α-globin genotype groupsRBC (x1012/L)MCH (pg)Hb (g/dL)Mentzer†RDWMCV (fL)group 1 (normal genotype)4,27±0,75 *25±1,5107±20 *17,4±2,4 *17,0±3,1 *76,6±3,2group 2 (single α-globin gene deletion)4,70±0,8525,2±1,5118±2216,2±2,9 *15,1±2,176,9±3,5group 3 (double α-globin gene deletion)5,20±0,5723,2±1,3 *120±914,1±2,1 *14,8±2,872,4±3,0 *† calculated for samples with Hb>90 g/L *indicates values that are significantly different from data of other groups. RBC: Red Blood Cells, MCH: Mean Corpuscular Hemoglobin, Hb: Hemoglobin, RDW: Red Cells Distribution Width, MCV: Mean Corpuscular Volume